Microvesicles are secreted by many, if not all, cell types in vitro and in vivo, and are present in biological fluids, such as, for example, blood, interstitial fluid, urine, saliva, and tears. Microvesicles are vesicles comprising lipid bilayers, formed from the plasma membrane of cells, and are heterogeneous in size, ranging from about 2 nm to about 5000 nm. The cell from which a microvesicle is formed is herein referred to as “the host cell”. Microvesicles are a heterogeneous population of vesicles and include, for example, ectosomes, microparticles, microvesicles, nanovesicles, shedding vesicles and membrane particles.
Microvesicles exhibit membrane proteins from their host cell on their membrane surface, and may also contain molecules within the microvesicle from the host cell, such as, for example, mRNA, miRNA, tRNA, RNA, DNA, lipids, proteins or infectious particles. These molecules may result from, or be, recombinant molecules introduced into the host cell. Microvesicles play a critical role in intercellular communication, and can act locally and distally within the body, inducing changes in cells by fusing with a target cell, introducing the molecules transported on and/or in the microvesicle to the target cell. For example, microvesicles have been implicated in anti-tumor reversal, cancer, tumor immune suppression, metastasis, tumor-stroma interactions, angiogenesis and tissue regeneration. Microvesicles may also be used to diagnose disease, as they have been shown to carry bio-markers of several diseases, including, for example, cardiac disease, HIV and leukemia.
Despite the importance of microvesicles, isolating microvesicles in useful quantities, while preserving their structural and functional integrity, remains problematic. The traditional procedure utilizes ultracentrifugation to isolate microvesicles from samples.
For example, U.S. Pat. No. 7,807,438 discloses a method for isolation of hepatitis C virus. The method comprises the separation of particles termed exosomes from the blood plasma of an individual infected with hepatitis C virus (HCV) and the extraction of RNA from these exosome particles.
In another example, U.S. Patent Application US20030198642A1 discloses [e]xosomes . . . derived from MHC class II enriched compartments in antigen presenting cells . . . [for] a . . . vaccination vehicle.
In another example, U.S. Patent Application US20060116321A1 discloses methods and compositions for use in mediating an immunosuppressive reaction. The compositions . . . comprise exosomes having immunosuppressive activity. Such exosomes may be derived from a variety of different cell types, including antigen-presenting cells such as dendritic cells and macrophages. Prior to isolation of exosomes, the cells may be genetically engineered to express molecules capable of enhancing the immunosuppressive activity of said exosomes and/or may be exposed to one or more agents, such as cytokines or cytokine inhibitors, which are also capable of enhancing the immunosuppressive activity of exosomes. The present invention also relates to the use of such exosomes for the treatment of diseases and disorders associated with undesirable activation of the immune system. The present invention also includes exosomes isolated directly from serum that have been shown to be immunosuppressive.
Ultracentrifugation may damage the microvesicles, resulting in the lysis or rupture of the vesicles. Damage to microvesicles may cause an adverse reaction in the body, if such damaged microvesicles were to be introduced.
Others have attempted alternate methods to isolate microvesicles. However, the alternate methods employed frequently isolate a sub-fraction of microvesicles, or are inefficient. For example, U.S. Patent Application US2011003008A1 discloses a particle secreted by a mesenchymal stem cell and comprising at least one biological property of a mesenchymal stem cell. The biological property may comprise a biological activity of a mesenchymal stem cell conditioned medium (MSC-CM) such as cardioprotection or reduction of infarct size. The particle may comprise a vesicle or an exosome.
In another example, U.S. Patent Application US20120070858A1 discloses a method for isolating exosomes from blood platelets using superparamagnetic nanoparticles of iron oxide (Fe3O4), by means of a charge attraction mechanism based on the predetermined Zeta potential of the exosomes. The method involves the use of iron oxide nanoparticles that are previously synthesised [sic] with a predetermined positive charge, and that bond to the negatively charged exosomes contained in the biological sample. During incubation, the cationic magnetic nanoparticles are absorbed by the surface of the membrane of the exosomes owing to electrostatic interaction. Exposure of the material to a magnetic field makes it possible to separate the exosomes bonded to the nanoparticles. The success of this technique has been confirmed by characterisation [sic] of the exosomes by flow citometry [sic]. The method has been shown to be suitable for this purpose, since it allows exosomes to be isolated and purified, without undergoing alterations of their original morphological and structural characteristics.
In another example, PCT Patent Application WO2012169970A1 discloses materials and methods for use of constrained cohydration agents in the purification of biological materials such as antibodies, viruses, cells, and cellular organelles in connection with convective chromatography, fluidized bed or co-precipitation applications.
There remains, therefore, a need to provide method to isolate and purify microvesicles without damage, and in sufficient quantities that the isolated microvesicles may subsequently be used for diagnosing disease, therapies, or research.
The present invention provides methods to isolate microvesicles from biological fluids without damaging the structural and/or functional integrity of the microvesicles. The present invention also provides methods to isolate ectosomes, microparticles, microvesicles, nanovesicles, shedding vesicles, apoptotic bodies, or membrane particles from biological fluids without damaging their structural and/or functional integrity.